Murine myeloid leukemia M1 cells undergo terminal differentiation to mature macrophages after stimulation with interleukin-6 (IL-6) and partial differentiation after stimulation with interferon gamma (IFNg). This cell line serves as a unique model for studying molecular and cellular events occuring during the process of myeloid differentiation. This system is being used to determine the dependency of expression of the enzyme nitric oxide synthase (NOS) on the stage of differentiation of macrophages. Nitric oxide (NO) is involved in bacterial and tumoricidal actions of macrophages. Regulation of NO production is controlled by an inducible form of NOS. This enzyme can be induced by exposure of macrophages to inflammatory stimuli such as lipopolysaccharide (LPS) and IFNg. The present study was undertaken to determine whether NOS induction is dependent on the stage of differentiation of the macrophage. When M1 cells were induced to differentiate with IL-6 NOS mRNA was detected 48 hours after induction and reached its peak of expression at 72 hours. The expression of lysozyme, a marker of macrophage differentiation, always preceded the expression of NOS. The expression of NOS was enhanced when IL-6-induced M1 cells were stimulated with IFNg for the final 6 hours before harvest of the cells. However, when IFNg is co-cultured with IL-6 for 72 hours, M1 cells undergo partial differentiation. Although M1 cells now express lysozyme the induction of NOS by IL-6 is inhibited by IFNg. Thus, the ability of macrophages to express NOS correlates with the maturation state of the macrophage.